DNA Microarray
DNA Microarray Human genomes include approximately 21,000 genes. All the cells in our bodies have some combination of these genes turned on and off. Scientists can sample cells and tissues by gene expression profiling via a technique called microarray analysis. This technique involves breaking open the cell, isolating its genetic components, and identifying all the genes that are turned on in that specific cell. Lists with all those genes are generated. DNA microarrays help scientists measure the expression level of numerous genes at the same time, as well as genotype multiple regions in a single genome. Scientists can tell if a gene is on if its mRNA is present. What is it? DNA microarrays, or DNA chips and biochips, are collections of microscopic DNA spots that are attached to solid surfaces. Each spot has multiple strands of specific DNA sequences, all of which are identical, and each spot represents one gene. Theses are called probes. Thousands of spots are arrayed in orderly rows and columns on solid surfaces, and the exact location of each spot is recorded for future reference. Probes can either be a short section of the gene or a part of another DNA element that can be used to hybridize cDNA and antisense RNA sample under high-stringency conditions. Targets that are labeled with fluorophore, silver and chemiluminescence are used to detect and quantify the abundance of nucleic acid sequences. How it Works Machines arrange small amounts of hundreds and thousands of gene sequences on a single microscope slide to create DNA microarrays. Scientists have access to a database that includes over 40,000 gene sequences that they can use to make the microarrays. The cellular machinery copies certain portions of the gene when it is activated, which creates a messenger RNA (mRNA). mRNA is the body’s template for creating proteins, and is complementary to the cell, which allows it to bind to the orginal DNA strand it was copied from. Scientists collects mRNA that is present in the cells they are studying and labels them using reverse transcriptase (RT) enzymes, which generate complementary cDNA to the mRNA. When this is happening, fluorescent nucleotides are attached to the cDNA. The tumor and normal samples are labeled with different fluorscent dyes. These samples are placed into a DNA microarray slide. The labeled cDNAs bind to their complementary DNAs on the slide and leave fluorescent tags. Special scanners measure the fluorescent intensity for each spot on the microarray slide. When genes are active, they have increased mRNA production and more labeled cDNA, which bind to the DNA on the slide and create a bright fluorescent area. Less active genes produce dimmer fluorescent spots. No fluorescence indicates that no messenger molecules are bound to DNA and that the gene is inactive. Scientists can test the activity of different genes at different times. When tumor samples are co-hybridized with normal samples, the tumor cells are dyed red and the normal cells are dyed green. The two complete for synthetic complementary DNAs on the slide. If the spot is red, the specific gene is more expressed in tumors that in normal cells and is up-regulated in cancer. If the spot is green, the gene is more expressed in normal tissue and is down-regulated in cancer. Yellow spots indicate that the gene is equally expressed in normal and tumor cells. Microarray Analysis and Cancer Microarray technology helps researchers learn more about different diseases. The study of cancer is intensely studied at the National Institutes of Health (NIH). Previously scientists were able to identify different kinds of cancers based on the organs the tumors were present in. However, microarray analysis identifies and categorizes different types of cancer based on the patterns of gene activity within the tumor cells. This allows researchers to design specific treatments that can target specific kinds of cancer. Furthermore, researchers are able to understand how different treatments affect the tumors by examining the gene activity between untreated and treated cells. This allows scientists to develop more effective treatments. References: http://en.wikipedia.org/wiki/DNA_microarray http://www.genome.gov/10000533 http://learn.genetics.utah.edu/content/labs/microarray/